Dendritic cells (DCs) initiate cell-mediated adaptive immune responses, with specialized DC subsets playing divergent roles in activation and differentiation of various effector T cells. Through the development of a novel microscopy methodology, Histo-Cytometry, we have recently learned that these subsets reside in different microanatomical regions in tissues. How DC subset positioning influences their capacity to respond to diverse immune perturbations and carry out their unique functions in vivo has not been determined. Also, which DCs normally mediate specific programs of T cell activation and differentiation and what factors regulate selective DC subset involvement in responses to differentially polarizing vaccines is not known. As these early priming events shape the outcome of cell-mediated immunity, we propose to utilize cutting edge microscopy techniques to directly in situ investigate how the spatial organization of DCs influences their ability to evoke T cell responses to distinct vaccines. Collectively, this work will lead to a better understanding of how the immune system is organized, which kind of cellular interactions are critical for functional immune responses, and how can we manipulate the immune system to improve disease outcomes.